Tunable Substrates
holes, rather than the surrounding carbon ( Figure 1 a and 1 b). T e same processes and coatings were then used to capture DLPs upon SiN microchips [ 7 ]. Surprisingly, the microchip specimens provided enhanced visual contrast in cryo-EM images ( Figure 1c ) in comparison to the holey carbon specimens. T ese insights provided the inspiration to develop tunable devices in which interchangeable adaptor molecules can be used to harvest healthy or dysfunctional proteins from human cancer cells for cryo-EM studies [ 8 , 9 ].
Figure 1 : Comparison of rotavirus assemblies prepared using different substrates and methods. (a) Rotavirus particles (black arrows) are primarily found in holes (white dashed circle) systematically engineered into carbon support fi lms that were plunge-frozen into liquid ethane for cryo-preservation. (b) Close-up view of rotavirus particles located in the holes of carbon support fi lm. (c) Image of rotavirus specimens prepared in the same manner and optimized on silicon nitride (SiN) [ 7 ].
(2) the breast cancer susceptibility protein (BRCA1); and (3) the BRCA1 binding partner, BARD1. T ese enriched components make up an active BRCA1-transcriptional complex that we were able to isolate using SiN microchips. Preparation of tunable microchips . To produce tunable SiN microchips, we used commercially available microchips (TEMwindows and Protochips, Inc.) that are hydrophobic in nature. T e microchips were decorated with a layered system that is used to capture specifi c complexes of interest. Microchips were coated with lipid monolayers containing Ni-NTA-functionalized phosphatidyl head groups (Avanti Polar Lipids) as previously described [ 7 – 9 ]. Briefl y, lipid stocks of both Ni-NTA lipids and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) were constituted to 1 mg/ml (w/v) in chloroform. T e Ni-NTA lipid allows for capture of the assemblies, while the DLPC lipid acts as a spacer. Lipid mixtures were cast over 15 μ L aliquots of Milli-Q water placed on parafi lm, then sealed in a petri dish and incubated on ice for 1 hour. For negatively stained specimens, 5% Ni-NTA lipid layers were used. For cryo-EM specimens, 25% Ni-NTA lipid layers were used, each of which also contained DLPC fi ller lipids (Avanti Polar Lipids). T e cleaned SiN microchips were placed on top of each monolayer droplet, and the microchips were gently liſt ed off of each droplet. T e coated microchips were incubated for 1 minute with aliquots (3 μ L each) of adaptor proteins including His-tagged protein A (AbCam) and IgG antibodies raised against the N-terminal (RING) or C-terminal (BRCT) domains of BRCA1. T e antibody-decorated microchips were then incubated for 2 minutes with aliquots of enriched nuclear material derived from breast cancer cells prior to plunge freezing [ 8 , 9 ].
Results
Using tunable substrates for cryo-EM applications . T e use of tunable substrates to prepare biological samples for cryo-EM analysis was recently demonstrated using Rotavirus double-layered particles (DLPs) as a model system. Applying functionalized coatings to holey carbon fi lms allowed us to observe a suffi cient number of virus particles in the desired
24
Figure 2 : BRCA1 assemblies formed in human cancer cells were isolated using SiN microchips. (a) Cancer cells were extracted, and the nuclear material was used to enrich for protein assemblies containing BRCA1. Enriched nuclear material was added to the microchip devices. (b) Microchip devices were coated with a lipid layer doped with Ni-NTA functionalized lipids. Tunable parts added to the devices included His-tagged protein A and antibodies for recruiting BRCA1 complexes. (c) Cryo-EM image of BRCA1-RNAP II assemblies poised on DNA [ 8 ].
www.microscopy-today.com • 2017 July
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